RNA interference (RNAi) is a cellular process that uses double stranded RNA (dsRNA) to target messenger RNA (mRNAs) for degradation and translation attenuation. The process is gene specific, refractory to small changes in target sequence, and amenable to multigene targeting. This phenomenon was first reported in plants in 1990 (Napoli, C., et al., Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell, 1990, 2(4):279-289). It was later observed in other organisms including fungi and worms (Romano, N., et al., Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol. Microbiol., 1992, 6(22):3343-53). Mechanistically, long dsRNA can be cleaved into short interfering RNA (siRNA) duplexes by Dicer, a Type III RNase. Subsequently, these small duplexes interact with the RNA Induced Silencing Complex (RISC), a multisubunit complex that contains both helicases and endonuclease activities that mediate degradation of homologous transcripts. The discovery that synthetic siRNAs of ˜19-29 bp can effectively inhibit gene expression in mammalian cells and animals has led to a flurry of activity to develop these inhibitors as therapeutics (Elbashir, S. M., et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411(6836):494-8). Recent development of advanced siRNA selection methods, including algorithm-based rational design selection, allows the researchers to select potent siRNA duplexes by key sequence and thermodynamic parameters that are target sequence independent (Khvorova, A., et al., Functional siRNAs and miRNAs exhibit strand bias. Cell, 2003, 115(1):209-216).
Small hairpin RNAs (shRNA) of 19-29 bp that are chemically synthesized or expressed from bacteriophage (e.g., T7, T3 or SP6) or mammalian (pol III such as U6 or H1 or pol II) promoters have also shown robust inhibition of target genes in mammalian cells. Furthermore, synthetic shRNA with its unimolecular structure has advantages in potency and simplicity over two-strand-comprising siRNA, the latter requiring the careful annealing of exact stoichiometric amounts of two separate strand which may have different purity profiles and off-target effects (Siolas, D., et al., Synthetic shRNAs as potent RNAi triggers. Nature Biotechnology, 2005, 23(2):227-231; Vlassov, A. V., et al., shRNAs targeting hepatitis C: effects of sequence and structural features, and comparison with siRNA. Oligonucleotides, 2007, 17:223-236).
However, a number of challenges have arisen over the course of shRNA design and development. First, researchers have observed wide-ranging variability in the level of silencing induced by different shRNA. Second, shRNA that varies in stem length, loop length and loop position has different knockdown capability (McManus, M. T., et al., Gene silencing using micro-RNA designed hairpins. RNA, 2002, 8:842-850; Harborth, J., et al., Sequence, chemical, and structural variatioin of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense And Nucleic Acid Drug Development. 2003, 13:83-105; Li, L., et al., Defining the optimal parameters for hairpin-based knockdown constructs. RNA, 2007, 13:1765-1774; Vlassov, A. V., et al., shRNAs targeting hepatitis C: effects of sequence and structural features, and comparison with siRNA. Oligonucleotides, 2007, 17:223-236). Some shRNAs having a 19-nucleotide guide strand at the 5′ end of the hairpin (left-hand loop) have been found to be more efficacious than those having their guide strand at the 3′ end of the hairpin (right-hand loop) (Scaringe, S. US 2004/0058886 A1), while other shRNAs were reported to be more efficacious with the right-hand loop structure (Vermeulen, et al. US 2006/0223777 A1). shRNAs with a 19-mer guide strand at the 3′ end of the hairpin (right-hand loop) were found to be more potent than 25- or 29-mer shRNAs (with right-hand loop). Previously available limited data suggested that the optimal loop size for a shRNA with a 19-bp stem with right-hand loop is larger than 4 nucleotides and preferably 9 or 10 nucleotides. A third challenge is that the mechanism of processing in the cytoplasm of synthetic shRNAs, especially 19-mer hairpins, is unknown. Unlike long dsRNAs and 29-mer shRNAs, 19-mer shRNAs are not cleaved by Dicer (Siolas, D., et al., Synthetic shRNAs as potent RNAi triggers. Nature Biotechnology, 2005, 23(2):227-231). Therefore, it would be advantageous to have a reliable design for superior shRNAs and a better understanding of the structure-activity relationship and processing of shRNAs to provide highly functional inhibitors.